Project Summary: The broad, long-term objective of this application is the development of a general method to reversibly regulate the stability (and thus function) of any protein of interest using synthetic, bioavailable small molecules. This application describes basic biomedical research that, if successful, would enable widespread advances in the diagnosis and treatment of human disease by facilitating fundamental biological studies as well as new animal models of human disease. It is known that fusion of an unstable protein domain to a second protein creates a chimeric protein that is unstable in cells and degraded by the proteasome. The goal of this application is to create an unstable protein domain that is conditionally stabilized when bound to a high-affinity ligand. For use in mice, the unstable domain would be fused to a protein of interest in the context of a knock- in mouse, and the stabilizing ligand would be constitutively administered until such time as it was desired to interrogate the role of the protein of interest. Withdrawal of the ligand would cause rapid degradation of the protein of interest. Readministering the ligand would stabilize the chimeric protein to normal levels. The specific aims of this application incorporate both rational design and diversity- based screening techniques to identify mutants of the FKBP protein that are unstable in mammalian cells but stable when bound to a cell-permeable, high-affinity ligand. New ligands that possess desirable pharmacological properties will be developed, and this method will be validated using several proteins of known scientific and therapeutic importance. Relevance: The goal of this research is to develop a general new method to rapidly and reversibly inactivate a specific protein in either cultured human cells or in mice. This methodology would enable the development of many new models for human diseases (e.g., cultured cells or knock-out mice), and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases.